1
13
TABLE 1. H and C NMR Spectral Data of Compound 1, J/Hz*
C atom
HMBC
C atom
HMBC
ꢁC
ꢁH
ꢁC
ꢁH
2
3
4
167.0 s
58.3 d
28.4 t
124.3 s
117.5 s
114.4 s
144.2 s
146.9 s
114.2 s
50.1 t
1ꢀ
2ꢀ
4.20 (1H, t, J = 7.0)
2.22 (1H, m)
1.91 (1H, m)
1.84 (2H, m)
3.39 (1H, m)
3.31 (1H, m)
4, 7
5,
2, 5
ꢀ
ꢀ
ꢀ
3
4
5
5
6
22.2 t
44.6 t
6ꢀ
7ꢀ
5.05 (1H, d, J = 15.0)
4.71 (1H, d, J = 15.0)
ꢀ
ꢀ
ꢀ
2, 9, 1 , 2 , 6
8
9
162.6 s
48.6 t
1ꢀ, 2ꢀ, 6ꢀ
3.92 (1H, d, J = 15.0)
3.17 (1H, d, J = 15.0)
8
2, 8
______
*Mutiplicities and coupling constants are in parentheses; assignment based on HMBC and HMQC experiments.
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The C NMR and DEPT spectra revealed the presence of 14 signals attributed to a diketopiperazine unit at ꢁ 167.0 (s, C-2),
C
58.3 (d, C-3), 28.4 (t, C-4), 22.2 (t, C-5), 44.6 (t, C-6), 162.6 (s, C-8), and 48.6 (t, C-9) which was composed of glycine and
proline and a 2,3,6-tribromo-4,5-dihydroxylbenzyl unit at 124.3 (s, C-1ꢀ), 117.5 (s, C-2ꢀ), 114.4 (s, C-3ꢀ), 144.2 (s, C-4ꢀ), 146.9
1
1
(s, C-5ꢀ), 114.2 (s, C-6ꢀ), and 50.1 (t, C-7ꢀ) [4]. The diketopiperazine unit was confirmed by the H– H COSY between H-3 and
H -4, H -4 and H -5, and H -5 and H -6 and the HMBC correlations from H-3 to C-4 and C-7, H-4b to C-2 and C-5, and
2
2
2
2
2
H-9b to C-2 and C-8. The 2,3,6-tribromo-4,5-dihydroxylbenzyl unit was confirmed by detailed comparison of the above NMR
data (Experimental section) with that of 1 in the literature [7] and the HMBC correlations from H-7ꢀa to 1ꢀ, 2ꢀ, and 6ꢀ. The C-N
bond was confirmed by the HMBC correlations from H -7ꢀ to 1ꢀ, 2ꢀ and 6ꢀ. Compound 1 was established as 2-(2,3,6-tribromo-
2
4,5-dihydroxybenzyl)hexahydropyrrolo[1,2-a]pyrazine-1,4-dione.
The configuration of proline was further confirmed by HPLC analyses of the acid hydrolysate of 1 after derivatization
with the Marfey reagent (FDAA), allowing the absolute configurations at the ꢂ-carbon to be assigned as the L-configuration
for proline [8].
EXPERIMENTAL
IR spectra were recorded by the FT-IR microscope transmission method on a Nicolet 5700 FT-IR spectrophotometer.
1
13
NMR spectra were recorded on a Varian Inova 500 MHz spectrometer at 500.103 MHz for H and 125.762 MHz for C in
DMSO-d using solvent signals (DMSO, ꢁ 2.50/ꢁ 39.5) as reference; the coupling constants are in Hz. HR-ESI-MS data
6
H
C
were measured with an APEX II FT-ICR-MS spectrometer (Bruker Daltonics, Inc. USA). Column chromatography was
performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc. China) and Sephadex LH-20 (Pharmacia Biotech AB,
Uppsala, Sweden). HPLC separation was performed on an Agilent 1100 series and an Alltima 250 cm ꢃ 2.2 cm i.d. preparative
column packed with C18 (10 ꢄm). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized
under UV light and by spraying with 2% FeCl in 95% EtOH.
3
Plant Material. Symphyocladia latiuscula was collected on the coast of Qingdao, Shandong Province, China in May
2004. The specimen was identified by Dr. Kui-Shuang Shao (Institute of Oceanology, ChineseAcademy of Sciences, Qingdao,
266071, China). A voucher specimen (No. 2004X16) was deposited at the Herbarium of the Institute of Oceanology, Chinese
Academy of Sciences, Qingdao, 266071, China.
Extraction and Isolation. The air-dried red alga Symphyocladia latiuscula (2.3 kg) was extracted with 95% EtOH at
room temperature for 3 ꢃ 72 h. After the solvent was removed under reduced pressure at < 40ꢅC, a dark residue (60 g) was
obtained. The residue was suspended in water and then partitioned with EtOAc. The EtOAc-soluble fraction (12 g) was
chromatographed over silica gel, eluting with a gradient of increasing Me CO (0–100%) in petroleum ether. The fraction
2
eluted by 30% Me CO in petroleum ether was rechromatographed over Sephadex LH-20 using petroleum ether–CHCl –MeOH
2
3
(5:5:1) to afford 18 subfractions. The ninth subfraction was further separated by reversed-phase preparative HPLC using
MeOH–H O–AcOH (75:20:0.1) as mobile phase to yield compound 1 (2.1 mg).
2
Absolute Configuration of Amino Acids [8]. Two set of compound 1 (50 ꢄg) were dissolved in 6 N HCl (400 ꢄL)
and put in 2 mL glass vials and heated at 110ꢅC for 24 h. Then the solvent was dried over nitrogen. The hydrolysate were
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